Transplantation of fatty livers is a major problem since many of them fail when used as donor organs. Because the source of liver grafts is largely accident victims who use alcohol, the relationship between fatty liver and graft failure needs to be understood to expand the pool of donor organs. Therefore, the goals of this project are to elucidate mechanisms involved in the failure of fatty livers and to develop strategies to prevent graft failure. Studies with chemicals are often not specific making interpretation difficult therefore. we will use mice with specific genetic deletions to test unique hypotheses about the source of free radicals and the role of endotoxin and TNFa in mechanisms of liver graft failure. Our first goal to be achieved in Aim 1 will be to optimize liver transplantation in knockout mice using treatments and techniques which are routine in this laboratory for rats. Recently, we have established liver transplantation here in the mouse. Survival and non-survival conditions will be established in the mouse, validating it as a useful model for mechanistic studies. Next, we will use knockout mice to provide unequivocal evidence for or against the hypothesis that endotoxin and TNFa play a critical role in failure of fatty grafts. To test this hypothesis, CD-14 knockout and TNF receptor I (TNFRI) knockout mice and appropriate wild-type controls will be treated with ethanol in Aim 2. The time course of survival, transaminase release and bile production will be observed after transplantation. NFKB (a pivotal transcription factor), TNFa and ICAM-1 will be measured at appropriate times after transplantation. We expect that deletion of CD-14 and TNFRI will improve survival of marginal livers. In Aim 3, the source of free radicals will be identified. Mitochondria in hepatocytes and NADPH oxidase in Kupffer cells are two potential sources of free radicals. Accordingly, knockouts of p47Phox, a required subunit of NADPH oxidase, and transgenic mice over expressing mitochondrial Mn-SOD will be given ethanol and livers will be transplanted under non-survival conditions. We expect that NADPH oxidase deficiency will prevent free radical production and reduce ethanol-induced primary non-function after liver transplantation. Depending on the results of Aims 2 & 3, targeted therapies such as ebselen, an antioxidant and anti-inflammatory drug, soluble TNFa receptor, and a novel endotoxin antagonist, E5331, will be evaluated in Aim 4. Taken together, we expect that by using the knockout approach these studies will provide solid evidence supporting the roles of endotoxin, free radicals, NFKB and TNFa in primary graft non-function in fatty livers. This work will develop mechanism-based strategies to increase the use of marginal fatty livers for transplantation in the clinic and save lives.